The Homology Analysis,transmission Mechanism And Genetic Environment Of NDM-5 Producing E.coli Isolates

Background and PurposeIn recent years,the excessive or unreasonable use of antibacterial drugs in the medical system,agriculture or industrial processes has led to the emergence and widespread spread of bacterial resistance.Due to the large investment and the long time in research and development of new antibacterial drugs,the frequency of use of broadspectrum antibiotic represented by carbapenem antibiotic has generally increased.Therefore,the induced bacteria resistance to these drugs is worrying.Carbapenemresistant Escherichia Coli(CREC),carbapenem-resistant Klebsiella pneumoniae(CRKP),carbapenem-resistant Acinetobacter baumannii(CRAB),carbapenemresistant Pseudomonas aeruginosa(CRPA),methicillin-resistant Staphylococcus aureus(MRSA),and penicillin-resistant Streptococcus pneumoniae(PRSP),such pandrug resistant,and even fully resistant bacteria continue to emerge,and the clinical antiinfection treatment situation is extremely severe.Carbapenem-resistant Escherichia Coli(CREC),as the most common and important member of carbapenem-resistant gram-negative bacteria,has attracted more attention.From a mechanism point of view,carrying NDM carbapenemase is the main reason for the widespread resistance of E.coli.Since British scientists first isolated the NDM-1 enzyme in New Delhi,India in 2007,28 NDM subtypes have been identified worldwide.Because NDM-carrying strains can hydrolyze almost all β-lactam antibiotics,the host bacteria are in a broadly resistant state.Currently,there are not many clinical treatments for infections caused by NDM-producing E.coli,only polymyxin and tigecycline can be temporarily controlled,and the mortality rate of patients is high.NDM-5 is a new type of carbapenemase that has emerged in recent years.Because of its wide hydrolysis spectrum of antibacterial drugs and strong resistance of host bacteria,it is one of the hotspots in the current research on the mechanism of bacterial resistance.We isolated 29 strains of E.coli carrying blaNDM-5 in a teaching hospital in Zhengzhou within five months in 2019,and the samples collected from blood,urine,sputum,anal swab,abdominal drainage,and bile and other parts,separated from the infection department,urology,ICU,gynecology,pediatrics,geriatric respiratory sleep department and other departments.This type of enzyme has a concentrated distribution of host bacteria,and there are many types of specimens and a wide range of sources,which has attracted our attention.Is there an outbreak of nosocomial infection in the host strain?What is the transfer mechanism of NDM-5 between bacteria?What is its genetic environment?We need to clarify these issues in order to facilitate clinical effective anti-infective treatments and provide reasonable guidance for curbing the spread of host bacteria and NDM-5 carbapenemase.Based on the above,we designed this subject to conduct a comprehensive analysis and research on the homology analysis,transmission mechanism,and genetic environment of the 29 isolated strains.Methods1.Collect 1587 non-repetitive strains continuously isolated from specimens from various departments of a teaching hospital in Zhengzhou within five months in 2019,use three-zone streaking to obtain a single colony,and use MALDI-TOF MS for bacterial species identification;use PCR amplification technology detects common carbapenem resistant genotypes(blaKPC,blaOXA-48,blaOXA-1,blaIIMP,blaNDM,blaOXA-10,blaVIM,MCR-1,MCR-2).Finally,29 strains of NDM-5 producing E.coli was included in this study.2.Extract the bacterial DNA and use the llumina HiSeq 4000-PE150 platform for next-generation sequencing,and use SPAdes 3.9.1 to sequence the data.Upload the obtained sequencing data to MLST(https://cge.cbs.dtu.dk/services/MLST/)to detect the ST typing of the strain.3.Use the kSNP program to identify whole-genome SNPs in the genome sequence.And run these SNPs to obtain a maximum likelihood tree of homology relationships between strains.The result is then labeled and decorated by iTOL.Search the information of strains carrying blaDNM-5 in the NCBI database.As of November 21,2020,a total of 1,839 strains of bacteria carrying blaNDM-5 were available in the NCBI database,of which 1310 were E.coli(TaxID 562).Of these,888 strains have uploaded data,of which 842 have ST types,and the data will be analyzed in this study with 29 strains carrying blaNDM-5 E.coli for phylogenetic analysis.Finally,the MLST and phylogenetic analysis results are combined to comprehensively analyze the strain homology.4.Use agar dilution method and broth micro-dilution method in vitro antibiotic susceptibility test to determine the characterization of bacterial resistance.5.The same use of the above second-generation sequencing results,using ResFinder 4.1(https://cge.cbs.dtu.dk/services/ResFinder/)to detect all antibiotic resistant genes carried by the strain,and analyze the whole genome information of the strain.6.Use S1 restriction enzyme digestion of PFGE,Southern blot hybridization,and Plasmidfinder program(https://cge.cbs.dtu.dk/services/PlasmidFinder)to explore the location of the antibiotic resistance gene and plasmid characteristics of the strain;use the plasmid conjugation experiment to detect whether the antibiotic resistance gene can be transfer to ascertain the spreading mechanism between bacteria.7.Upload sequencing data to NCBI,and download its annotation data through Prokaryotic Genome Annotation Pipeline(PGAP)for the accuracy of antibioticresistant genes and elements positioning.8.Use Easyfig program to generate a linear genetic environment map,and annotate the antibiotic-resistant genes and various originals in the genetic environment map based on the above-mentioned annotation data.Results1.From April to October in 2019,1,587 strains were collected from various departments of a teaching hospital in Zhengzhou.29 NDM-5 carbapenemase-producing E.coli strains were selected and included in this study.2.MLST results showed that 8 different ST types were detected in 29 strains of NDM-5 producing E.coli,including 10 strains of ST167,4 strains of ST361,5 strains of ST405,5 strains of ST410,1 strain of ST744,2 strains of ST746,1 strain of ST4948 and 1 strain of ST11179.Among them,ST11179 is the first type discovered in the world.Among these strains,ST 167 is the most common type.3.According to phylogenetic analysis,ST167 is a high-risk clone of NDM-5 producing E.coli,ST405,ST617,ST410 and ST648 are the common ST types,among which ST167 strains and ST617 strains show a higher genetic relationship.Each ST type is very extensive in source type and country distribution.The source types include blood,urine,sputum,feces,pus,secretions,bile,pleural and ascites,drainage fluid,etc.The countries of separation include China,the United States,Pakistan,Myanmar,Israel,India and so on.In addition,the tested strains ZDHYF423 and ZDHYF595,ZDHYF689 and ZDHYF769,ZDHYF796 and ZDHYS737 were all isolated from the same patient in the study.Phylogenetic analysis found that the SNP-distances of ZDHYF423 and ZDHYF595,ZDHYF689 and ZDHYF769 are 0,which means that the strains ZDHYF423 and ZDHYF595,ZDHYF689 and ZDHYF769 are homologous strains,while ZDHYF423 and ZDHYF595 are ST361 type,and ZDHYF689 and ZDHYF769 type ST167.This shows that ST361 and ST167 have clonal transmission in this study.The ST typing of other strains is diverse,which means that the spread of the blaNDM-5 resistance gene in this study is not only caused by the spread of the cloned strain.The SNP-distances of ZDHYF796 and ZDHYS737 is 1,which indicates that our patient’s infection strain has mutated or re-infected with non-homologous strains.In addition,the antibiotic resistance of the three groups of strains is different,which shows that the antibiotic resistance has changed under the action of antibacterial drugs.4.Antibiotic susceptibility results showed that 29 isolated bacteria in this study were all multi-antibiotic resistant bacteria,and 85%of the strains were resistant or intermediate to cefotaxime,ceftazidime,cefepime,imipenem,meropenem,gentamicin,chloramphenicol,amoxicillin-clavulanic acid,piperacillin-tazobactam,aztreonam,levofloxacin and ciprofloxacin.5.29 strains of NDM-5 producing E.coli carry a variety of antibiotic resistance genes,including β-lactams,macrolides,carbapenems,quinolones,sulfonamides,aminoglycosides,tigecycline,fosfomycin,amide alcohols,rifampicin and trimethoprim.F147,F322,F514,F752,S255,S258,S363,S365,S373 not only carry the blaNDM-5 carbapenem resistance gene,but also carry the blaOXA-1 carbapenem resistance gene.The macrolide resistance gene mdfA was detected in all strains.6.In this study,the blaNDM-5 genes carried by 29 E.coli strains were all located on transferable plasmid.The blaNDM-5 gene is located on IncX3,IncFII,IncFIA and other plasmid types,mainly located on the～40 kb IncX3 plasmid.There are 10 types of genetic environment surrounding the blaNDM-5 gene,all of which have conservative sequences:blaNDM5-bleMBL-trpF-dsbC.Conclusion1.ST167 is a high-risk clone of NDM-5 producing E,coli in this study,and the first discovery named ST11179,suggesting that the strain has a new mutation,and the antibiotic resistance of the mutant strain will also change.2.The clonal spread of resistance genes between strains is a way to mediate the spread of blaNDM-5.The self-transfer of IncX3 plasmid is also a key factor in promoting the spread of resistance genes.3.The gene environment of blaNDM-5 is diverse,but all have blaNDM-5-bleMBL-trpFdsbC conservative sequences.